Abstract. Complementary to efforts supported by this grant that led to the characterization of the first signaling fatty acid primary amide, its hydrolysis and signaling termination by and discovery of fatty acid amide hydrolase (FAAH), and the validation of FAAH as a therapeutic target modulating the activity of endocannabinoids, we will target additional serine hydrolases that regulate the release or degradation of these key signaling fatty acid- derived endocannabinoids (i.e., anandamide and 2-arachidonylglycerol (2-AG)). With the concerns surrounding the use of COX-2 inhibitors for the treatment of chronic pain, the liabilities of opioids (respiratory depression, desensitization with chronic dosing, dependence, constipation), and the challenges of promoting direct cannabinoid use (catalepsy, hypomotility, hypothermia, dependence), interest in therapeutic targeting of the endocannabinoid system is now intense. Rather than blunt force targeting of the signaling receptors (CB1 and CB2), the modulation of the release or termination of the endocannabinoid signaling molecules has emerged as an especially attractive therapeutic approach that avoids cannabinoid side effects by only potentiating active signaling at their needed sites of action. This development is largely the result of our studies and those of our collaborators with whom we discovered and characterized FAAH, and for which we provided the first inhibitors that were sufficiently efficacious in vivo to validate the target for the treatment of pain without the characteristic cannabinoid or opioid side effects. Our continued efforts will define therapeutic opportunities for the treatment of pain and inflammatory disorders by using inhibitors of additional enzymes regulating the signaling of endocannabinoids (anandamide and 2-AG), targeting dual FAAH/MAGL inhibitors, selective MAGL inhibitors, and selective DAGL-? inhibitors. In these studies we will continue to create targeted screening libraries for this major enzyme class and use activity-based protein profiling (ABPP) to screen for potent, selective inhibitors of uncharacterized serine hydrolases for use in identifying their endogenous roles and potential to serve as therapeutic targets. Central to the science to emerge from our work was the discovery of the presence, and subsequently the role, of fatty acid amide signaling molecules. Studies continuing to define the site of action and endogenous role of such fatty acid amide signaling molecules will be conducted, including those targeting erucamide and arachidonamide.